SIGNAL TRANSDUCTION IN INTERFERON-INDUCED TRANSCRIPTION

干扰素诱导转录中的信号转导

• 批准号: 2064717
• 负责人: David E Levy
• 金额: $ 20.11万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 1994-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2064717
• 关键词: DNA binding protein; HeLa cells; Lagomorpha; Xenopus; affinity chromatography; alternatives to animals in research; antibody formation; antibody receptor; antiserum; bacterial proteins; biological signal transduction; cell growth regulation; communicable disease control; complementary DNA; gene complementation; gene expression; genetic regulatory element; genetic transcription; genetic translation; interferons; ligands; membrane proteins; molecular cloning; molecular pathology; nucleic acid sequence; oligonucleotides; peptide hormone; protein purification; protein signal sequence; receptor; receptor binding; site directed mutagenesis; tissue /cell culture; transcription factor; transposon /insertion element; virus infection mechanism

The physiological state of a cell is modulated in response to specific extracellular signals through changes in metabolic processes resulting, in part, from differential regulation of gene expression. The broad objective of this proposal is to characterize the molecular mechanisms involved in signal transduction initiated by polypeptide ligands binding to their specific cell surface receptors. Interferons (IFNs) area a class of polypeptide cytokines which induce rapid and coordinate transcriptional activation of a set of dispersed interferon stimulated genes (ISGs), leading to profound effects on the growth, differentiation, and metabolism of sensitive cells. The activation of these genes is completely dependent on the presence of IFN bound to its cell surface receptor and is mediated through the action of pre-existing protein components which change in activity, rather than in abundance, following treatment with IFN. Although the cis-acting DNA sequences regulating these genes have been characterized and trans-acting nuclear and cytoplasmic factors have been identified, the protein components of the signal transduction pathway from the cell surface to the nucleus, the nature of the biochemical events which carry the signal, and the mechanisms of transcriptional control of ISGs remain at present largely undefined. The major goal of this project is to develop a comprehensive understanding of IFN-induced transcription which leads to production of gene products necessary for growth control and for viral resistance. We propose to determine the molecular mechanics of IFN-induced signal transduction by defining genes and gene products in the pathway, by purifying and characterizing constituent proteins, and by defining the biochemical steps involved in signal generation. In this proposal, the individual components of signal transduction will be isolated and purified in order to reconstitute the process in vitro and to study the biochemical steps involved. This will be approached by isolating, purifying, and cloning the positive transcription factor which activates ISG expression in the nucleus, the protein precursors to this transcription factor which exists in the cytoplasm, the proteins which interact with these components to achieve activation and nuclear translocation, the type I IFN cell surface receptor which initially generates the signal, and membrane and cytoplasmic receptor-proximal proteins which serve as links in the signal chain. These proteins will be analyzed in vitro and in vivo by mutagenesis to define functional domains involved in IFN binding, signal transduction, subunit association, nuclear translocation, DNA binding, and transcriptional activation. Knowledge of the normal mechanism of action of IFN will provide a basis for understanding its role in viral inhibition and control of proliferation and will lead to investigations of defects in these processes which may contribute to impaired resistance to infectious disease and to abnormal cell growth. These studies may also be broadly applicable signal transduction systems involving polypeptide hormones and growth factors.

细胞的生理状态根据特定的条件进行调节 通过代谢过程的变化产生细胞外信号， 部分来自基因表达的差异调节。 广泛的目标 该提案的目的是描述参与的分子机制 由与其结合的多肽配体启动的信号转导 特定的细胞表面受体。 干扰素 (IFN) 属于一类 诱导快速协调转录的多肽细胞因子 激活一组分散的干扰素刺激基因（ISG）， 对生长、分化和新陈代谢产生深远影响 敏感细胞。 这些基因的激活完全依赖于 干扰素的存在与其细胞表面受体结合并介导 通过预先存在的蛋白质成分的作用，这些成分发生变化 IFN 治疗后活性，而不是大量。 虽然 调节这些基因的顺式作用 DNA 序列已得到表征 反式作用核因子和细胞质因子已被鉴定， 细胞表面信号转导途径的蛋白质成分 对于细胞核来说，生化事件的本质是 信号，并且ISGs的转录控制机制仍然存在 目前基本上未定义。 该项目的主要目标是形成全面的了解 IFN 诱导的转录导致基因产物的产生 生长控制和病毒抵抗所必需的。 我们建议 确定 IFN 诱导信号转导的分子机制 通过纯化和定义途径中的基因和基因产物 表征组成蛋白，并定义生化步骤 参与信号产生。 在此提案中，各个组件 的信号转导将被分离和纯化，以便 体外重建该过程并研究生化步骤 涉及。 这将通过分离、纯化和克隆来实现 激活ISG表达的正转录因子 细胞核，存在该转录因子的蛋白质前体 在细胞质中，与这些成分相互作用的蛋白质 实现细胞表面I型IFN的激活和核转位 最初产生信号的受体，膜和细胞质 近受体蛋白，充当信号链中的链接。 这些 蛋白质将通过诱变进行体外和体内分析，以确定 参与 IFN 结合、信号转导、亚基的功能域 关联、核易位、DNA 结合和转录 激活。 了解干扰素的正常作用机制将为以下方面提供基础： 了解其在病毒抑制和增殖控制中的作用 将导致对这些过程中的缺陷进行调查，这可能 导致对传染病的抵抗力受损并导致异常 细胞生长。 这些研究也可能是广泛适用的信号 涉及多肽激素和生长因子的转导系统。